Cytotoxicity on human cells of Cry1Ab and Cry1Ac Bt

J
Journal Code
A T
2
Article ID
Dispatch: 10.12.11
7 1 2 No. of Pages: 5
CE:
ME:
Short Communication
Q1
Received: 5 May 2011,
Revised: 15 November 2011,
Accepted: 19 November 2011
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jat.2712
Cytotoxicity on human cells of Cry1Ab and
Cry1Ac Bt insecticidal toxins alone or with a
glyphosate-based herbicide
R. Mesnage,a,b E. Clair,a,b S. Gress,a,b C. Then,c A. Székácsd and
G.-E. Séralinia,b*
ABSTRACT: The study of combined effects of pesticides represents a challenge for toxicology. In the case of the new growing
generation of genetically modified (GM) plants with stacked traits, glyphosate-based herbicides (like Roundup) residues are
present in the Roundup-tolerant edible plants (especially corns) and mixed with modified Bt insecticidal toxins that are produced by the GM plants themselves. The potential side effects of these combined pesticides on human cells are investigated
in this work. Here we have tested for the very first time Cry1Ab and Cry1Ac Bt toxins (10 ppb to 100 ppm) on the human
embryonic kidney cell line 293, as well as their combined actions with Roundup, within 24 h, on three biomarkers of cell death:
measurements of mitochondrial succinate dehydrogenase, adenylate kinase release by membrane alterations and caspase 3/7
inductions. Cry1Ab caused cell death from 100 ppm. For Cry1Ac, under such conditions, no effects were detected. The Roundup
tested alone from 1 to 20 000 ppm is necrotic and apoptotic from 50 ppm, far below agricultural dilutions (50% lethal concentration 57.5 ppm). The only measured significant combined effect was that Cry1Ab and Cry1Ac reduced caspases 3/7 activations
induced by Roundup; this could delay the activation of apoptosis. There was the same tendency for the other markers. In these
results, we argue that modified Bt toxins are not inert on nontarget human cells, and that they can present combined sideeffects with other residues of pesticides specific to GM plants. Copyright © 2011 John Wiley & Sons, Ltd.
Keywords: Roundup; Bt toxins; Cry1Ab; Cry1Ac; GMOs; mixtures; glyphosate; human cells
INTRODUCTION
The real effects of mixtures of chemical pollutants are a major
concern for public health (Monosson, 2005). Humans are
exposed to hundreds of compounds on a daily basis. The commercialized combinations could be a first matter of concern.
Agricultural genetically modified organisms (GMOs) are steadily
increasing worldwide, and they need to be carefully assessed
(Séralini et al., 2009, 2011; Spiroux de Vendômois et al., 2010).
Nowadays 99.9% of GMOs can be described as pesticide plants,
designed for herbicide tolerance and/or modified insecticide
production (James, 2010). Thus pesticides residues co-occur in
the plant, synthesized by the plant itself, by the expression of
the inserted transgene (modified Bt from Bacillus thuringiensis)
or through external pesticide treatment facilitated by the transgene-dependent tolerance to herbicides (Roundup in most
instances). In turn, such residues exert their effects upon consumption or release into the environment (Arregui et al, 2004;
Tank et al., 2010). Owing to their key role in intensive agriculture,
potential side effects of such combined pesticides residues
should be assessed. In vitro tests are frequently recommended
as a first step to replace animal models in toxicity studies. Here,
we have tested for the first time the effects of Cry1Ab and
Cry1Ac alone and combined with Roundup on human cells.
Modified toxins from Bt are Cry proteins forming pores in
insect cell membranes (Then, 2010); they account for 39% of
edible plant GMOs worldwide (James, 2010). Since natural Bt toxins
have long been used, their modified counterparts are often compared with them. However, the latter derivatives are truncated,
J. Appl. Toxicol. 2011
adapted and modified synthetic sequences; consequently their
activity is possibly quite different from the natural ones (Séralini
et al., 2011). Also, Bt toxins are claimed and believed to be safe.
Yet prions, hormones and venoms are also proteins, and are far
from being innocuous. To date, Bt toxins have not been tested on
human cells. However, Bt corns are regularly consumed by humans
in America and their residues have even been found in maternal
and fetal cord serum at around 0.2 ppb (Aris and Leblanc, 2011),
which does not take into account the tissue levels. Nontarget toxicity of natural Bt toxins has been detected in mammals, for instance
at a 50% lethal concentration (LC50) from around 10 to 520 ppb (Ito
et al., 2004; Nagamatsu et al., 2010; Rani and Balaraman, 1996).
Roundup formulations are mixtures of glyphosate and adjuvants such as ammonium sulfate, benzisothiazolone, glycerine,
isobutane, isopropylamine, polyethoxylated alkylamines and
*Correspondence to: G.-E. Séralini, University of Caen, Risk Pole MRSH-CNRS,
Laboratory of Biochemistry EA2608, Esplanade de la Paix, 14032 Caen cedex, France.
E-mail: criigen@unicaen.fr
a
University of Caen, Risk Pole MRSH-CNRS, Laboratory of Biochemistry EA2608,
Esplanade de la Paix, 14032 Caen cedex, France
b
CRIIGEN, 40 rue de Monceau, 75008 Paris, France
c
Testbiotech e.V., Frohschammerstr. 14, 80807 München, Germany
d
Plant Protection Institute, Hungarian Academy of Sciences, H-1525, Budapest,
POB 102, Hungary
Copyright © 2011 John Wiley & Sons, Ltd.
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sorbic acid (Cox, 2004). Glyphosate-based herbicides are the
object of an increasing number of studies, which reveal, in
combination with adjuvants, endocrine-disrupting effects, and
tumor-promoting or teratogenic effects on numerous nontarget
species (Gasnier et al., 2009; George et al., 2010; Paganelli et al.,
2010), for instance from 0.5 ppm on androgen receptors. We
have used human embryonic kidney cell line HEK293 as a sensitive model (Benachour and Séralini, 2009). The kidney model
was used also because a body of evidence suggests kidney dysfunctions as endpoints of GMO diet effects (Séralini et al., 2011),
and thus kidney cells could be a target for GMOs. We first measured the mitochondrial respiration level, by succinate dehydrogenase (SD) activity assessment in order to test cytotoxicity.
Then, as Bt proteins act as pore forming toxins (Then, 2010),
we determined adenylate kinase (AK) activity when released in
the medium, revealing possible membrane alterations. In association, we assayed caspase 3 and 7 activities in order to separate
the apoptotic and necrotic actions involved in cytotoxic effects.
Moreover, human cell lines allow the study of unintended side
effects on nontarget species of GMO-associated pesticides.
MATERIALS AND METHODS
Chemicals
Cry1Ab and Cry1Ac were prepared as described previously by
two different laboratories (Székács et al, 2010; Pusztai-Carey
et al., 1994). The glyphosate-based herbicide tested was commercially available RoundupW GT Plus formulation, approval
no. 2020448 (Monsanto, Anvers, Belgium). It contains 450 g l 1
glyphosate acid (N-phosphonomethyl-glycine). Successive dilutions were prepared in Eagle’s modified minimum essential
medium (EMEM; Abcys, Paris, France). 3-(4,5-Dimethyl-thiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) and all other compounds
were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France),
unless specified. MTT was prepared as a 5 mg ml 1 stock solution in
phosphate-buffered saline, filtered through a 0.22 mm filter before
use, and diluted to 1 mg ml 1 in EMEM.
Toxin Preparations
The Cry1Ab and Cry1Ac toxins are cloned from the natural
Bacillus thuringiensis subspecies kurstaki HD-1 strain and
expressed in Escherichia coli as single gene products. The inclusion bodies, containing the protoxins, were solubilized at pH
10.5 in the presence of b-mercaptoethanol and treated with
commercial bovine trypsin (Sigma, USA). The 65 kDa activated
toxins were isolated by ion exchange HPLC and the pure toxin
fractions were desalted and lyophilized and stored at 80 C.
After storage, toxins were diluted in a 50 mM Na-carbonate–HCl
buffer at 1 mg ml 1 (pH 9.5), and then diluted in EMEM.
Cell Lines
The human embryonic kidney 293 cell line (ECACC 85120602)
was provided by Sigma-Aldrich (Saint-Quentin Fallavier, France).
Cells were grown in phenol red-free EMEM (Abcys, Paris, France)
containing 2 mM glutamine, 1% nonessential amino acid,
100 U ml 1 of antibiotics (a mixture of penicillin, streptomycin
and fungizone; Lonza, Saint Beauzire, France), 10 mg ml 1 of liquid
kanamycin (Dominique Dutscher, Brumath, France) and 10% fetal
bovine serum (PAA, les Mureaux, France). Cells were grown at
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37 C (5% CO2, 95% air) during 24 h to 80% confluence, washed
with serum-free EMEM and then exposed to various chemicals,
since the serum delayed the cell necrosis by about 48 h in the
presence of toxic compounds (Benachour et al., 2007). The
control cells grow normally in serum-free medium up to 96 h.
Cell Treatments and Cytotoxicity Biomarkers
Cells at 80% confluence in 48- or 96-well plates (Dominique
Dutscher, Brumath, France) were washed with serum-free EMEM,
in order to avoid other combined effects, and then exposed to
various concentrations of Bt toxins or Roundup GT Plus in EMEM
serum-free medium for 24 h. Bt toxins were used from 10 ppb to
100 ppm (in the range of GM plant production). Concerning
Roundup, 50% lethal concentrations (LC50) were assessed from
1 to 20 000 ppm (the latter is the agricultural dilution). Combined
effects were measured by mixing LC50 of Roundup with three
doses of each Bt toxin. After treatments, the following tests were
applied: mitochondrial respiration assay (MTT) through the succinate dehydrogenase activity measurement (Mosmann, 1983).
The optical density was measured at 570 nm using a Mithras LB
940 luminometer (Berthold, Thoiry, France). The bioluminescent
ToxiLight bioassay (Lonza, Saint Beauzire, France) was applied for
the membrane degradation assessment, by the intracellular AK
release in the medium; this is described as a necrosis marker
(Crouch et al., 1993). Finally, the apoptotic cell death was evaluated
with the Caspase-Glo 3/7 assay (Promega, Paris, France). Luminescence was measured using a Mithras LB 940 luminometer (Berthold,
Thoiry, France). These methods were previously described by our
group (Benachour and Séralini, 2009).
Statistical Analysis
The experiments were repeated at least three times in different
weeks on three independent cultures (n = 9). LC50 values were
calculated by a nonlinear regression using a sigmoid (fiveparameter) equation with the GraphPad Prism 5 software. All data
were presented as the means standard errors (SEs). Statistical
differences were determined by Student t-test using significance
levels at P < 0.01 (**) and P < 0.05 (*).
RESULTS
We measured for the first time cytotoxic effects of Bt toxins,
alone or in combination with a glyphosate-based herbicide, on
HEK293 cells. First of all, we confirmed that the buffer was not
cytototoxic for the cells. The mitochondrial succinate dehydrogenase activity of treated cells significantly decreased at
100 ppm of Cry1Ab alone (by 11%, Fig. 1A). Even if it was limited,
this phenomenon was undetected for Cry1Ac. Lower doses were
tested from 10 ppb to 10 ppm, but significant effects were not
observed. We obtained similar effects with a Cry1Ab toxin provided by another laboratory that was prepared and stored independently (Fig. 1A). We measured AK activity after its release in
the medium in order to evaluate plasma membrane integrity.
A concentration of Cry1Ab of 100 ppm increased AK leakage in
the medium 2-fold, revealing plasma membrane alterations. This
was performed for the two Cry1Ab toxins. Apoptotic effects of
Cry1Ab and Cry1Ac by means of caspase 3/7 activities were studied; no effects on HEK293 cells were visible. We can therefore
confirm that Cry1Ab can induce cytotoxic effects via a necrotic
mechanism in these conditions at 100 ppm.
Copyright © 2011 John Wiley & Sons, Ltd.
J. Appl. Toxicol. 2011
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Cytotoxicity of Cry1Ab and Cry1Ac Bt insecticidal toxins
Figure 1. Cytotoxic effects of modified Bt toxins Cry1Ac and Cry1Ab on
HEK293 cell line. (A) Cell death has been measured on mitochondrial
succinate dehydrogenase after 24 h exposure to two Cry1Ab toxins up
to 100 ppm prepared from different sources (A, black curves with circles
and squares) and Cry1Ac (grey curve). (B) Cell membrane degradation
was measured by adenylate kinase release (RU, relative units) provoked
by Cry1Ab and Cry1Ac toxins (1–100 ppm) in comparison to control (C).
No effect was detected on caspase 3/7 activities; therefore results are
not displayed. Standard errors of the mean are indicated in all instances
(n = 9; * P < 0.05).
Closer to the reality of exposure, we then tested combined
effects of Bt toxins with Roundup (Fig 2). According to our
previous results, Roundup is cytotoxic by inhibition of mitochondrial respiration activity, far below agricultural dilutions
(around 200 times less) with an LC50 of 57.5 ppm (Fig. 2A).
At this concentration, Roundup also induced necrosis
evidenced by a 15-fold increase of an AK release. Apoptosis
induction was measured by a 6.7-fold caspase 3/7 activities
enhancement (Fig. 2B). However, concerning combined effects
we observed significant effects on apoptosis; both Bt toxins
from 10 ppm reduced caspase 3/7 activities (by around 50%)
when they were induced in Roundup at its LC50 (Fig. 2B).
Similarly, there was a non significant tendency for both toxins
(data not shown) to reduce AK leakage and mitochondrial respiration inhibition induced by Roundup.
DISCUSSION
Few studies have been performed on nontarget effects of Bt
toxins, and none with modified Bt toxins extracted from plants,
or together with Roundup residues, even in regulatory files. For
natural Bt toxins, their mechanisms of action and insect resistance are not fully understood (Singh and Sivaprasad, 2009),
and the metabolism of these proteins in mammals is unknown
(Séralini et al., 2011; Chowdhury et al., 2003). They may even
interact with extrinsic factors (Then, 2010). Billions of people
and wildlife could be exposed to modified Bt toxins; therefore
understanding their potential side effects is crucial.
J. Appl. Toxicol. 2011
Figure 2. Effects of Roundup alone and with modified Bt toxins Cry1Ab
and Cry1Ac on HEK293 cell line. (A) Cells were treated with Roundup
(1–20 000 ppm) and the mitochondrial respiration was measured
through succinate dehydrogenase activity (left). The LC50 for Roundup
was determined as 57.5 ppm by nonlinear regression (arrow). On the
right, the adenylate kinase release showing membrane alterations by
Roundup alone at its LC50 in comparison to control. (B) Combined effects
of Roundup at its LC50 with Bt toxins measured on caspase 3/7 activities
(%). The same tendency for the combined effects but with no significant
results was detected on adenylate kinase release and succinate dehydrogenase; results not shown. Standards errors of the mean are indicated in
all instances (n = 9). Significance of the effects (* P < 0.05; ** P < 0.01) is
tested against the negative control C (medium alone) or the positive
Roundup control (R).
On two biomarkers of cell death, Cry1Ab exposure led to
respiration inhibition and plasma membrane alterations, by contrast to Cry1Ac. This could be consistent with the fact that the
consumption of MON810 maize producing Cry1Ab (in the ppm
range) induced signs of hepatorenal alterations in a subchronic
feeding study on rats (Spiroux de Vendômois et al., 2009). It is
known that both toxins differ significantly in their domain III
structure (Karim and Dean, 2000), which is the only one to be
involved at the same time in ion channel function, receptor
binding and insertion into the membrane (Dean et al., 1996).
This occurred at relatively high concentrations (100 ppm)
in comparison to the concentrations produced in GM plants
(1–20 ppm, Székács et al., 2010). The content can differ greatly
according to the GM variety and environmental conditions
(Then and Lorch, 2008). The exposure during consumption can
appear low enough to avoid side effects, and whether this
occurs in vivo remains to be checked. However, the bioaccumulation in tissues, or bioaccumulative or long-term effects, has to
be taken into account since Bt residues were recently claimed to
be measured in pregnant women’s serum at around 0.2 ppb (Aris
and Leblanc, 2011). In addition, high quantities of Bt crops can
be consumed by mammals. The procedure for GMO market
authorizations for crops such as MON810 (EFSA, 2009) does
not require in vitro tests on human cells of Bt toxins, nor on its
combinatorial effects, thus our results are raising new questions
about the safety of these toxins and the Bt crops in general.
Although in vitro studies suggest degradation in human gastric
Copyright © 2011 John Wiley & Sons, Ltd.
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Acknowledgments
We are grateful to Testbiotech and the GEKKO Foundation, the
association Denis Guichard, CRIIGEN and the Conseil Regional
of Basse-Normandie for their support. R.M. and E.C. are recipients
of fellowships from CRIIGEN and the Conseil Regional of BasseNormandie. We would also like to thank Marianne Pusztai-Carey
for one of the toxin preparations.
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secretions, digestion is never a complete process and insecticide
toxins cannot be fully degraded in vivo (Paul et al., 2010). This is
known to be the case for Cry1Ab (Chowdhury et al., 2003). It
must be underlined that the insecticidal proteins produced by
the GM plants are in soluble forms, and thus already biochemically activated, unlike those produced by the microorganism
Bt, secreted as inactive precursors or protoxins (Hilbeck and
Schmidt, 2006). The importance of Bt toxin activation has been
demonstrated in relation to in vitro membrane damages of human erythrocytes, by solubilized Bt toxins, but not by the intact
form (Rani and Balaraman, 1996). Cellular response to Bt toxins
does not elicit apoptosis; it induces necrotic effects via a plasma
membrane disruption for Cry1Ab within only 24 h. This may be
due to pore formation like in insect cells owing to binding to
specific receptors or membrane lipid rafts (Then, 2010; Soberón
et al., 2009).
We also demonstrated that Cry1Ab and Cry1Ac exposures
slightly reduced caspase 3/7 activations induced by Roundup.
This could be related at least in part to the properties of
Roundup compounds, especially adjuvants. We observed previously, in our group, that serum delayed the cytotoxic effects
induced by Roundup. This was probably due to serum binding
proteins (Benachour et al., 2007). Here we can assume that physico-chemical properties of proteins may give them the ability
to bind and form complexes with Roundup adjuvants that have
tendencies to form vesicles, buffering their bioavailability to
cells. Similarly, a nonsignificant tendency of reduction of the
cytotoxic effects of Roundup was observed on mitochondrial
respiration and membrane degradation when the toxins were
added. The apoptosis induction appeared to be the most
sensitive impact of combined effects. This does not exclude
other intracellular targets such as endocrine disruption, since
Roundup is antiandrogenic from 0.5 ppm, below toxic levels
and close to human serum levels (0.1–0.2 ppm in Acquavella
et al., 2004).
Here we documented that modified Bt toxins are not inert on
human cells, but can exert toxicity, at least under certain in vitro
conditions. In vivo implications should be now assessed. Our
results raise new questions in the risk assessment of food and
feed derived from genetically engineered plants.
wileyonlinelibrary.com/journal/jat
Copyright © 2011 John Wiley & Sons, Ltd.
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J. Appl. Toxicol. 2011 130
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How much Bt toxin do genetically engineered MON810 maize plants
actually produce? Theor. Ökol. 14: 1–5.
Copyright © 2011 John Wiley & Sons, Ltd.
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